The present invention relates to a process for producing a coenzyme Q10 for pharmaceutical and other uses. More particularly, the invention relates to a process for producing coenzyme Q10 which comprises isolating a gene coding for the coenzyme Q10 side-chain synthase, which is a key enzyme involved in the biosynthesis of coenzyme Q10, i.e. decaprenyl diphosphate synthase, from a fungal strain of the genus Saitoella and introducing it into a host microorganism to let it elaborate coenzyme Q10.
The conventional technology for commercial production of coenzyme Q10 comprises isolating the coenzyme from a tobacco or other plant and modifying the length of its side chain by a synthetic technique.
While it is known that coenzyme Q10 is produced by a broad spectrum of organisms ranging from microorganisms, such as bacteria and yeasts, to higher animals and plants, the method comprising culturing a microorganism and extracting coenzyme Q10 from the microorganism is regarded as one of the most effective production methods and has actually been exploited commercially. However, the prior art methods are invariably poor in productivity, providing for only low outputs and/or involving time-consuming procedures.
The pathways for biosynthesis of coenzyme Q10 in organisms are partly different between the prokaryote and the eukaryote but invariably comprise a complicated cascade of reactions involving many kinds of enzymes. However, these pathways are basically comprised of three fundamental steps, namely the step of synthesizing decaprenyl diphosphate as the precursor of the prenyl side-chain of coenzyme Q10, the step of synthesizing p-hydroxybenzoic acid as the basis of the quinone ring of coenzyme Q10, and the step of coupling these two compounds together and effecting a serial substituent transformation to complete coenzyme Q10. Of these reactions, the reaction determinant of the length of the side-chain of coenzyme Q10 and acknowledged to be the rate-determining step of its biosynthesis, i.e. the reaction catalyzed by decaprenyl diphosphate synthase, is considered to be the most important reaction. Therefore, in order that coenzyme Q10 may be produced with good efficiency, it seems worthwhile to isolate the key gene involved in said biosynthesis, namely the gene coding for decaprenyl diphosphate synthase, and utilize it for enhanced production of the enzyme. As sources of the gene, fungi capable of producing coenzyme Q10 in comparatively large amounts can be regarded as useful candidates.
Heretofore, genes coding for decaprenyl diphosphate synthase have been isolated from several kinds of microorganisms, such as Schizosacch aromyces pombe (JP09-173076A) and Gluconobacter suboxydans (JP10-57072A), etc., but the inherent coenzyme Q10 productivity of these micrcorganisms cannot be considered high enough and neither an efficient cultural protocol for these microorganisms nor an efficient isolation and purification procedure has been established as yet. Therefore, there has been a standing demand for isolation of a coenzyme Q10-encoding gene from a microorganism capable of highly producing a coenzyme Q10.
Devoted to providing a solution to the above-mentioned production problems, the present invention has for its object to isolate a gene coding for the enzyme synthesizing the coenzyme Q10 side chain from a fungal strain of the genus Saitoella and exploit it to advantage for the efficient microbial production of coenzyme Q10.
To accomplish the above object, in the present invention, the key gene involved in the biosynthesis of coenzyme Q10, namely the gene coding for decaprenyl diphosphate synthase, was isolated from a fungal strain of the genus Saitoella in the first place. Then, this gene was introduced and allowed to be expressed in a host microorganism, such as Escherichia coli, to thereby enable the host to produce coenzyme Q10 with efficiency.
The inventors of the present invention made intensive investigations for isolating such genes coding for decaprenyl diphosphate synthase from fungal strains of the genus Saitoella capable of producing comparatively large amounts of coenzyme Q10 and have succeeded in isolating said genes.
The present invention, therefore, is concerned with a DNA of the following (a), (b) or (c).
(a) a DNA having the nucleotide sequence shown under SEQ ID NO:1
(b) a DNA having a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:1 by the deletion, addition, insertion and/or substitution of one or a plurality of nucleotides
and coding for a protein having decaprenyl diphosphate synthase activity
(c) a DNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO:1 under a stringent condition and codes for a protein having decaprenyl diphosphate synthase activity.
The present invention is further concerned with a protein of the following (d) or (e).
(d) a protein having the amino acid sequence shown under SEQ ID NO:2
(e) a protein having an amino acid sequence derived from the amino acid sequence of SEQ ID NO:2 by the deletion, addition, insertion and/or substitution of one or a plurality of amino acids
and having decaprenyl diphosphate synthase activity.
The invention is further concerned with a DNA coding for this protein.
The present invention is further concerned with an expression vector containing said DNA. For the expression vector of the invention, various vector systems heretofore known can be utilized and, therefore, may for example be pNTSal as constructed by cloning the DNA having the sequence of SEQ ID NO:1 into the vector pUCNT for expression.
The present invention is further concerned with a transformant as constructed by transforming a host microorganism with said DNA. As the host microorganism for the invention, Escherichia coli can be used with advantage.
The invention is further concerned with a process for producing coenzyme Q10 
which comprises culturing said transformant in a culture broth and harvesting the coenzyme Q10 produced and accumulated in the resulting culture. The host microorganism for this process is not particularly restricted but may be Escherichia coli to mention a preferred example. The coenzyme Q produced by Escherichia coli is coenzyme Q8 but the invention enables this microorganism to produce coenzyme Q10.
The inventors made intensive investigations on the isolation of the enzyme gene from a fungal strain which belongs to the genus Saitoella and is capable of producing comparatively large amounts of coenzyme Q10 and succeeded in acquiring a fragment of the particular gene by a PCR technique.
The inventors compared the sequence of the known gene coding for decaprenyl diphosphate synthase with the genes cording for polyprenyl diphosphate synthases, namely long-chain prenyl synthases which are analogous to said known enzyme gene but differ from the same in chain length and, for the region of high homology, synthesized various PCR primers. Using these primers in various combinations, they studied PCR conditions. As a result, they found by analysis of the gene sequence that when a PCR using DPS-1 (SEQ ID NO:3) and DPS-1 1AS (SEQ ID NO:4) as primers is carried out according to the protocol of heat-treatment at 94xc2x0 C.xc3x973 minutes, followed by 40 cycles of 94xc2x0 C., 1 minutexe2x86x9243xc2x0 C., 2 minxe2x86x9272xc2x0 C., 2 minutes, a ca 220 bp fragment of the enzyme gene can be amplified from the chromosome gene of Saitoella complicata IFO 10748, a fungus belonging to the genus Saitoella.
Then, to acquire the full length of this enzyme gene, the chromosome gene Saitoella complicata IFO 10748 is digested with the restriction enzyme EcoRI and inserted into a xcex phage vector to construct a recombinant phage library. After the plaque is transferred to a nylon membrane, the plaque hybridization is carried out using the labeled PCR fragment, whereby a clone having the full-length decaprenyl diphosphate synthase gene can be obtained.
Sequencing of the decaprenyl diphosphate synthase gene occurring in the above clone reveals that the gene has the nucleotide sequence shown under SEQ ID NO:1 of SEQUENCE LISTING. The amino acid sequence deduced from the above nucleotide sequence is shown under SEQ ID NO:2. Here, a sequence characteristic of a gene coding for decaprenyl diphosphate synthase is observed.
The DNA of the invention may be any of the DNA having the nucleotide sequence shown under SEQ ID NO:1, the DNA having a nucleotide sequence derived from the sequence of SEQ ID NO:1 by the deletion, addition, insertion and/or substitution of one or a plurality of nucleotides and coding for a protein having decaprenyl diphosphate synthase activity, and the DNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO:1 under a stringent condition and codes for a protein having decaprenyl diphosphate synthase activity.
The xe2x80x9cnucleotide sequence derived by the deletion, addition, insertion and/or substitution of one or a plurality of nucleotidesxe2x80x9d means any nucleotide sequence derived by the deletion, addition, insertion and/or substitution of a number of nucleotides of the order which can be deleted, added, inserted and/or substituted by the methods well known in the art, for example as described in, inter alia, Protein, Nucleic Acid, Enzyme, Supplemental Issue: Gene Amplification PCR Technology TAKKAJ 35 (17), 2951-3178 (1990) and Henry A. Erlich (ed.), PCR Technology (the translation edited by Ikunoshin Kato) (1990).
As used in this specification, the term xe2x80x9cprotein having decaprenyl diphosphate synthase activityxe2x80x9d means a protein capable of synthesizing decaprenyl diphosphate in a yield of not less than 10%, preferably not less than 40%, more preferably not less than 60%, still more preferably not less than 80%, relative to the protein having the amino acid sequence shown under SEQ ID NO:2. Such yield measurements can be made by the technique which comprises reacting FDP (farnesyl diphosphate) and 14C-IPP (radiolabeled isopentenyl diphosphate) with the enzyme of interest, hydrolyzing the resulting 14C-DPP (decaprenyl diphosphate) with phosphatase, fractionating the hydrolysate by TLC, and assaying the amounts taken up in spots corresponding to the respective chain lengths (Okada et al., Eur. J. Biochem., 255, 55 to 59).
The xe2x80x9cDNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO:1 under a stringent conditionxe2x80x9d means a DNA obtained by colony hybridization, plaque hybridization, Southern hybridization or the like hybridization technique using the DNA having the nucleotide sequence of SEQ ID NO:1 as the probe. Anyone skilled in the art may easily acquire the objective DNA by carrying out said hybridization according to the methods described in Molecular Cloning, 2nd Edition (Cold Spring Harbor Laboratory Press, 1989).
The protein of the present invention may have the amino acid sequence shown under SEQ ID NO:2 or an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:2 by the deletion, addition, insertion and/or substitution of one or a plurality of amino acids and having decaprenyl diphosphate synthase activity.
xe2x80x9cThe amino acid sequence derived by the deletion, addition, insertion and/or substitution of one or a plurality of amino acidsxe2x80x9d can be obtained by effecting such deletion, addition, insertion and/or substitution by the technology well known in the art, such as a region-specific mutagenesis technique. Specific procedures are described in Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983) and other literature.
The protein of the present invention preferably has an amino acid sequence showing a homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90% further still more preferably not less than 95%, to the amino acid sequence shown under SEQ ID NO:2.
The xe2x80x9chomologyxe2x80x9d is calculated by aligning two nucleotide sequences to be compared in the optimum format, counting the matched base positions (A, T, C, G, U or I) between the two sequences, dividing the count by the total number of bases compared, and multiplying the product by 100. Specifically, this calculation can be made using an analytical software such as Hitachi Soft Engineering""s DNASIS, Software Development""s GENETYX, or Finland CSC""s Clustal X, for instance.
While the gene coding for decaprenyl diphosphate synthase must be ligated downstream of a suitable promoter for expression, an expression vector can be constructed, for example by excising a DNA fragment containing the gene with a restriction enzyme or amplifying the enzyme-encoding gene selectively by PCR, followed by cloning it into a vector having a promoter. In the present invention, the expression vector into which the DNA coding for the protein having decaprenyl diphosphate synthase activity may be inserted is not particularly restricted but may for example be one constructed by ligating a suitable promoter to a plasmid derived from E. coli. The plasmid of E. coli origin includes pBR322, pBR325, pUC19 and pUC119, while the promoter includes T7 promoter, trp promoter, tac promoter, lac promoter and xcexPL promoter. Further, as the expression vector of this invention, pGEX-2T, pGEX-3T, pGEX-3X (all from Pharmacia), pBluescript, pUC19 (from Toyobo), pMALC2, pET-3T and pUCNT (described in WO 94/03613), etc. can also be mentioned. Among these, pUCNT can be used with advantage. To mention a specific example, the vector pNTSal for the expression of a decaprenyl diphosphate synthase gene can be constructed by inserting the gene having the DNA sequence shown under SEQ ID NO:1 into the expression vector pUCNT.
Then, this enzyme gene expression vector is introduced into a suitable microorganism, whereby the microorganism is rendered capable of producing coenzyme Q10. The host microorganism is not particularly restricted but Escherichia coli can be used with advantage. The Escherichia coli is not particularly restricted but includes such strains as XL1-Blue, BL-21, JM109, NM522, DH5xcex1, HB101 and DH5, among others. Among these, E. coli DH5xcex1 can be used with particular advantage. For example, when the expression vector pNTSal containing the decaprenyl diphosphate synthase gene is introduced into this E. coli strain, the coenzyme Q10, which the intact E. coli inherently does not produce, can be produced in a large amount. This E. coli DH5xcex1 (pNTSal) has been deposited with National Institute of Bioscience and Human-Technology (Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) under the accession number of FERM BP-6844.
Furthermore, Escherichia coli KO229 (Journal of Bacteriology, 179, 3058-3060 (1997), the octaprenyl diphosphate synthase gene-knockout E. coli strain constructed by Kawamukai et al. as the host microorganism, is incapable of producing coenzyme Q8 and can be utilized as the host for higher production of coenzyme Q10.
The gene can be used not only singly but may be introduced together with another biosynthesis-related gene into a microorganism to thereby obtain still more satisfactory results.
Coenzyme Q10 can be produced by culturing the transformant obtained according to the invention and harvesting the product coenzyme Q10 in a per se known manner. When the host microorganism is a strain of Escherichia coli, either LB broth or M9 broth containing glucose and casamino acids can be used as the culture broth. In order that the promoter may be allowed to function with efficiency, the broth may be supplemented with ascertain chemical such as isopropyl-thiogalactoside or indolyl-3-acrylic acid. Culture can be carried out at 37xc2x0 C. for 17 to 24 hours, for instance, optionally under aeration or agitation. In the practice of the invention, the product coenzyme Q10 may be used after purification or as it is in the crude form, depending on the intended use. Isolation of coenzyme Q10 from the culture can be made by using known separation and purification procedures in a suitable combination. As such known separation and purification procedures, there can be mentioned techniques utilizing solubilities, such as salting-out and solvent precipitation; techniques chiefly utilizing differences in molecular weight, such as dialysis, ultrafiltration, gel filtration and (SDS-)polyacrylamide gel electrophoresis; techniques utilizing differences in charge, such as ion exchange chromatography; techniques utilizing specific affinity, such as affinity chromatography; techniques utilizing differences in hydrophobicity, such as reversed-phase high performance liquid chromatography; and techniques utilizing differences in isoelectric point, such as isoelectric focusing, among others.
The use for the coenzyme Q10 obtained according to the invention is not particularly restricted but the enzyme can be applied to pharmaceuticals with advantage.